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Serum Cystatin C for Renal Function Assessment

A New Particle-Enhanced Turbidimetric Assay



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July 2011

Introduction

Cystatin C is an alternative blood biomarker of kidney function.1,2 This small protein (molecular weight 13,250 Daltons) is produced by nucleated cells and is freely filtered by the glomerulus. Blood levels are not greatly influenced by age, gender, or body size. Therefore, cystatin C has attracted interest as an alternative marker of glomerular filtration rate (GFR). Much effort has been expended to standardize serum creatinine measurement.3 However, relatively little effort has been devoted to standardize cystatin C measurement until recently, when the International Federation of Clinical Chemistry (IFCC) formed a working group to produce an international reference material (ERM-DA471/IFCC) which was released in June 2010. Recently, Mayo Clinic’s Renal Lab evaluated a new cystatin C particle-enhanced turbidimetric assay (PETIA) (Gentian AS, Moss, Norway) that is traceable to this certified cystatin C reference material4 and can be performed on a routine chemistry analyzer.

Particle-Enhanced Turbidimetric Assay Performance

Analytic performance of the new PETIA assay is excellent. Within-run and within-lab imprecision is low, with coefficient of variations (CV) of 0.65% to 1.33% for cystatin C levels in the most common clinical range between 0.98 and 1.88 mg/L5 (reference range=0.57-1.29 mg/L). The lower limit of quantitation is 0.35 mg/L, with an analytic measurement range up to 6 mg/L. This platform affords the advantages of higher throughput, increased availability (more easily adapted to 24/7 lab operation), shorter turnaround time, and lower cost.6

Comparison With Current Nephelometric Assay

A total of 142 samples were run on the new PETIA and previous Siemens (formerly Dade Behring) particle-enhanced nephelometric immunoassay (PENIA). An unexpected observation was a consistent 23% bias across the measured range with the PETIA showing higher results (Figure 1). Therefore, certified IFCC cystatin C reference material was obtained to further compare the 2 assays. At an expected cystatin C value of 1.38 mg/L, the PETIA measured a cystatin C concentration of 1.35 mg/L (98% of expected). However, the corresponding PENIA result was only 1.10 mg/L (80% recovery). Thus, the PETIA, but not the PENIA, yielded expected results for this international reference material.

Figure 1

Figure 1. Comparison of cystatin C values obtained by PENIA and PETIA assays. A 23% bias was observed that was consistent across the measured range.

Recent reports suggest that drift had occured in the Siemens cystatin C assay between 2006 and 2010.7 To determine if a similar drift in Mayo Clinic’s PENIA assay over time might explain the discrepancy between the current PENIA and PETIA results, biobanked samples from a reference value study conducted at Mayo Clinic in 2000 using the Dade Behring (now Siemens) PENIA were reanalyzed on the same platform. The 2000 PENIA results were 19% higher across the measurement range when compared to values obtained in 2010 (Figure 2), and differed in a direction that could account for much of the current assay discrepancy observed between the PENIA and PETIA results. Furthermore, when a subset of the biobanked samples (n=40) were performed on the new platform, PETIA results were 25% higher than the PENIA results, suggesting the fall in PENIA results over time was not the consequence of sample deterioration. Since the PETIA is traceable to an international reference material, while the Siemens PENIA is not, we suspect that the values from the PETIA are more likely to be accurate. These studies highlight the importance of standardization if cystatin C is to be employed to estimate GFR.

Comparison With Creatinine-Based Estimated Glomerular Filtration Rate

Serum cystatin C values obtained using the PETIA were used to estimate GFR using a published formula developed using this assay:5

estimated GFR=77.24 x (cystatin C mg/L)-1.2623

We studied 100 patients that were undergoing clinically indicated GFR testing by iothalamate renal clearance at Mayo Clinic. In general, the cystatin-based equation performed reasonably well with an overall -7.4% bias for all patients analyzed. When broken down by category, for the 2 largest subgroups, known chronic kidney disease (CKD) patients and potential kidney donors (no known kidney disease), the bias was -14.5% and +7.2%, respectively. For comparison, GFR was also estimated from concurrent serum creatinine values and the widely used Modification of Diet in Renal Disease (MDRD) equation.8 In general, the MDRD equation did not perform as well as the cystatin-based equation, showing an overall -12.9% bias compared to measured GFR. When analyzed for CKD and potential kidney donors, the bias of MDRD-estimated GFR compared to measured GFR was -10% and -20.6%, respectively. The sensitivity and specificity of each equation for detecting a GFR <30, <60, and <80 was also assessed, as well as the percentage of estimated GFR values that fell within 30% of the measured GFR result (Table). In general, the PETIA cystatin C formula performed best, with good sensitivity and specificity across the GFR range of the samples we studied.

Figure 2

Figure 2. Cystatin C PENIA results on biobanked samples analyzed in 2000 and again in 2010. A 19% bias was observed that was constant across the measurement range (results in 2000 higher than in 2010).


 
GFR (mL per minute per 1.73 m²)
% within 30%
of measured
GFR
<30
<60
<80
Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity
PETIA CC GFR 100% 92% 94% 79% 94% 88% 79%
MDRD eGFR 96% 96% 96% 77% 97% 63% 78%

Table. Sensitivity and specificity of each equation by GFR level
Abbreviations: PETIA, particle-enhanced turbidimetric assay; CC, cystatin C; eGFR, estimated glomerular filtration rate; MDRD, modification of diet in renal disease; GFR, glomerular filtration rate

Discussion

Cystatin C is a low-molecular–weight cysteine proteinase inhibitor that is produced by all nucleated cells and found in body fluids, including serum. Since it appears to be formed at a constant rate and freely filtered by the kidneys, its serum concentration is inversely correlated with GFR; that is, high values indicate low GFRs (eg, renal disease, decreased renal perfusion) while lower values indicate higher GFRs, similar to creatinine. Unlike creatinine, cystatin C production is not related to muscle mass. Therefore, blood levels of cystatin C may be a better marker for GFR than creatinine.1 However, several recent large studies have developed equations that can partially overcome the known weakness of creatinine by using demographic criteria (age, race, gender) to model the relationship between serum creatinine and GFR.8 Use of estimated GFR represents a marked improvement over serum creatinine alone, and widespread implementation has negated some of the urgency for wide adaptation of cystatin C as an alternative biomarker of GFR. Indeed, recent studies have demonstrated that cystatin C levels may differ in patients with and without CKD, and in relationship to other clinical features such as transplant status.5

Renal Anatomy and  Filteration

Renal anatomy and filtration. Cystatin C is freely filtered in the glomerulus to pass from blood into tubular fluid. As this filtrate travels through the nephron to the collecting duct and drains into the renal pelvis and ureter, cystatin C is neither secreted nor reabsorbed. Therefore, clearance of cystatin C is a good marker of GFR. Furthermore, since cystatin C is produced at a constant rate in the body, blood levels are a good indicator of GFR.

Like serum creatinine, formulas have been developed to take demographics into account and estimate GFR from cystatin C levels. These formulas perform at least as well as those based on creatinine to estimate GFR, and in certain circumstances (eg, patients with abnormal muscle mass) may be better. Furthermore, cystatin C appears to be a strong prognostic indicator for further cardiovascular events, mortality, and kidney failure.9 Therefore, it has been proposed that cystatin C could serve as a “tier 2” test to confirm CKD among patients with reduced estimated GFR by creatinine-based equations.9 Cystatin C may also have a role for estimating GFR among hospitalized patients in order to detect acute kidney injury earlier and more accurately dose renally excreted drugs,2 or as a cardiovascular prognostic indicator.9 It may also be useful when GFR is in the so-called “creatinine clearance blind zone” of 50 to 90 mL/min/1.73 m2 where estimates of GFR using serum creatinine can produce falsely low numbers if the patient is not known to have CKD.10

Facts About Chronic Kidney Disease (CKD)
  • 26 million American adults have CKD and millions of others are at increased risk.
  • High-risk groups for CKD include those with diabetes, hypertension, and a family history of kidney disease.
  • African Americans, Hispanics, Pacific Islanders, Native Americans and seniors are also at increased risk for CKD.
  • Glomerular filtration rate (GFR) is the single best estimate of kidney function.
  • Persistent proteinuria (increased protein in the urine) also means CKD is present.
  • Early detection can help prevent the progression of early CKD to total kidney failure.
  • It is important to control blood pressure since hypertension causes CKD and CKD causes hypertension.
  • Heart disease is the major cause of death for all people with CKD.
Blood levels of cystatin C are a good marker of GFR because it is:
  • Generated at a constant rate largely independent of age, gender, and size
  • Freely filtered by the glomerulus
  • Not secreted by renal tubules

Conclusion

The recent studies performed by Mayo Clinic’s Renal Function Laboratory detailed in this article indicate that cystatin C can be accurately measured by PETIA on a routine chemistry analyzer, and then used to estimate GFR with existing equations. Advantages of this assay over the previously offered PENIA include improved turnaround time and standardization to international reference material. The analytic performance characteristics (accuracy by split samples, reportable range, and precision) and day-to-day performance are robust, even among operators. Our studies provide some evidence that cystatin C-based estimated GFR may have better sensitivity and specificity than creatinine-based estimated GFR for detecting CKD, especially if the GFR is in the range that is not detected by changes in creatinine and in patients with abnormal muscle mass.

Authored by John Lieske, MD and Nikolay Voskoboev

Dr. John Lieske, Medical Director of Mayo Clinic Renal Testing Laboratory in Rochester, Minnesota, also presents a Hot Topic recording – Use of Cystatin C to Assess Kidney Function – which will be available on August 1, 2011. Please visit our Web site (www.mayomedicallaboratories.com) to view.

References

  1. Westhuyzen J. Cystatin C: a promising marker and predictor of impaired renal function. Ann Clin Lab Sci 2006;36:387-394
  2. Nejat M, Pickering JW, Walker RJ, Endre ZH. Rapid detection of acute kidney injury by plasma cystatin C in the intensive care unit. Nephrol Dial Transplant 2010;25:3283-3289
  3. Myers GL, Miller WG, Coresh J, et al: Recommendations for improving serum creatinine measurement: a report from the Laboratory Working Group of the National Kidney Disease Education Program. Clin Chem 2006;52:5-18
  4. Blirup-Jensen S, Grubb A, Lindstrom V, et al: Standardization of Cystatin C: development of primary and secondary reference preparations. Scand J Clin Lab Invest Suppl 2008;241:67-70
  5. Rule AD, Bergstralh EJ, Slezak JM, et al: Glomerular filtration rate estimated by cystatin C among different clinical presentations. Kidney Int 2006;69:399-405
  6. Flodin M, Jonsson AS, Hansson LO, et al: Evaluation of Gentian cystatin C reagent on Abbott Ci8200 and calculation of glomerular filtration rate expressed in mL/min/1.73 m(2) from the cystatin C values in mg/L. Scand J Clin Lab Invest 2007;67:560-567
  7. Larsson A, Hansson LO, Flodin M, et al: Calibration of the Siemens Cystatin C Immunoassay Has Changed Over Time. Clin Chem 2011;56:777-778
  8. Levey AS, Bosch JP, Lewis JB, et al: A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med 1999;130:461-470
  9. Peralta CA, Katz R, Sarnak MJ, et al: Cystatin C identifies chronic kidney disease patients at higher risk for complications. J Am Soc Nephrol 2011;22:147-155
  10. Rule AD, Larson TS, Bergstralh EJ, Slezak JM, et al: Using serum creatinine to estimate glomerular filtration rate: accuracy in good health and in chronic kidney disease. Ann Intern Med 2004;141:929-937

 


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